Yale Scientists Find Marker for High-Quality Induced Pluripotent Stem Cells

Pluripotent stem cells can be made by genetically engineering adult cells into less mature cells that have pluripotency. These induced pluripotent stem cells or iPSCs can potentially differentiate into any cell type in the adult body and because they are made from the patient’s own cells, they have a lower risk of being rejected by the patient’s immune system.

However, iPSCs suffer from an increased mutation rate when they are made and these increased mutation rate increases their risk of causing tumors and being rejected by the patient’s immune system. Having said that, not all iPSCs are created equal, and the safety of iPSCs seems to be very line-specific. Thus, how do you know a good stem cell from a bad one?

Yale Stem Cell Center researchers led by Andrew Xiao Yale have published a report in the Sept. 4 issue of Cell Stem Cell in which they describe an indicator that seems to predict which batch of personalized stem cells will differentiate into patient-specific tissue types and which will develop into unusable placental or tumor-like tissues.

Xiao’s group identified a variant histone protein called H2A.X that seems to predict the developmental path of iPSC cells in mice. Histone proteins assemble into tiny spools around which DNA winds. This DNA spooling allows cells to tightly package their DNA into a tight, compact structure that is easily stored called “chromatin.” Histones that are commonly used include histones H2A, H2B, H3 and H4.

Core histones

Two copies of each of these proteins assemble into a globular structure called a core histone and the DNA of the cell winds around this core histone to form a “nucleosome.” Then linker histones (H1 or H5) take these nucleosomes package them into spiraled coils.

DNA solenoids

H2A.X is a variant version of histone H2A is modified when DNA damage occurs. Modified H2A.X signals to the DNA repair machinery to fix the broken DNA (see TT Paull, and others, Curr. Biol. 10(15):886–95).


According to the data from Xiao’s research team, in pluripotent stem cells, H2A.X is specifically targeted to those genes typically expressed in cells used to make the placenta, and it helps suppress differentiation of pluripotent stem cells into cells of the placental lineage. Given this distribution in mouse embryonic stem cells, H2A.X deposition pattern is a functional marker of the quality of iPSCs. Conversely, defective H2A.X deposition predisposes iPSCs toward differentiating into placental-type cells and tumors.

“The trend is to raise the standards and quality very high, so we can think about using these cells in clinic,” Xiao said. “With our assay, we have a reliable molecular marker that can tell what is a good cell and what is a bad one.”

Increasing Reprogramming Efficiency by Turning Off One Gene

The removal of one genetic roadblock could improve the efficiency of adult cell reprogramming by some 10 to 30 fold, according to research by stem cell scientists at the Methodist Hospital Research Institute and two other institutions.

Rongfu Wang, the principal investigator and director of the Center for Inflammation and Epigenetics, said this about his group’s findings: “The discovery six years ago that scientist can convert adult cells into inducible pluripotent stem cells, or iPSCs, bolstered the dream that a patient’s own cells might be reprogrammed to make patient-specific iPSCs for regenerative medicine, modeling human diseases in Petri dishes, and drug screening. But reprogramming efficiency has remained very low, impeding its applications in the clinic.”

Wang and his group identified a protein encoded by a gene called Jmjd3, which is known as KDM6B, acts as an impediment to the reprogramming of adult cells into iPSCs. Jmjd3 is involved in several different biological processes, including the maturation of nerve cells and immune cell differentiation (Popov N, Gil J. Epigenetics. 2010 5(8):685-90).

These findings by Wang’s team are the first time anyone has identified a role for Jmjd3 in the reprogramming process. According to Wang, fibroblasts that lack functional Jmjd3 showed greatly enhanced reprogramming efficiency.

Helen Wang, one of the co-principal authors of this study, said, “Our findings demonstrate a previously unrecognized role of Jmjd3 in cellular reprogramming and provide molecular insight into the mechanisms by which the Jmjd3-PHF20 axis controls this process.’

While teasing apart the roles of Jmjd3 in reprogramming, Wang and his colleagues discovered that this protein regulates cell growth and cellular aging. These are two previously unidentified functions of Jmjd3, and Jmjd3 appears to work primarily by inactivating the protein PHF20. PHF20 is a protein that is required for adult cell reprogramming, and cells that lack PHF20 do not undergo reprogramming to iPSCs.

Rongfu Want explained it like this: “So when it comes to increasing iPSC yields, knocking down Jmjd3 is like hitting two birds with one stone.”

Jmjd3 is almost certainly not the only genetic roadblock to stem cell conversion. Wang noted, “Removal of multiple roadblacks could further enhance the reprogramming efficiency with which researchers can efficiently generate patient-specific iPSCs for clinical applications.”

While this is certainly an exciting finding, there is almost certainly a caveat that comes with it. increased reprogramming efficiency almost certainly brings the potential for increased numbers of mutations. Other studies have shown that iPSC generation is much more efficient if the protein P53 is inhibited, but P53 is the guardian of the genome. It prevents the cell from dividing if there is substantial amounts of DNA damage. Inhibiting P53 activity allows iPSC generation even if the cells have excessive amounts of DNA damage. Therefore, inhibiting those cellular processes that are meant to guard against excessive cell proliferation and growth can lead to greater numbers of mutations. Thus, before Jmjd3 inactivation is used to generate iPSCs for clinical uses, extensive animal testing must be required to ensure that this procedure does make iPSCs even less safe than they already are.

What Does Breast Cancer Have to Do With Skin Stem Cells?

BRCA1 is a gene that plays a huge role in breast cancer. Particular mutations in BRCA1 predispose women increased risks of breast cancer cervical, uterine, pancreatic, and colon cancer and men to increased risks of pancreatic cancer, testicular cancer, and early-onset prostate cancer.

BRCA1 encodes a protein that helps repair damage to chromosomes. When this protein product does not function properly, cells cannot properly repair acquired chromosomal damage, and they die or become transformed into cancer cells.

What does this have to do with stem cells? A study led by Cédric Blanpain from the Université libre de Bruxelles showed that BRCA1 is critical for the maintenance of hair follicle stem cells.

Peggy Sotiropoulou and her colleagues in Blanpain’s laboratory showed that when BRCA1 is deleted, hair follicle cells how very high levels of DNA damage and cell death. This accumulated DNA damaged drives the follicle stem cells to divide furiously until they burn themselves out. This is in contrast to the other stem cell populations in the skin, particularly those in the sebaceous glands and epidermis, which are maintained and seem unaffected by deletion of BRCA1.

Sotiropoulou said of these results: “We were very surprised to see that distinct types of cells residing within the same tissue may exhibit such profoundly different responses to the deletion of the same crucial gene for DNA repair.”

This work provides some of the first clues about how DNA repair mechanisms in different types of adult stem cells are employed at different stages of stem cells activation. Blanpain and his group is determining if other stem cells in the body are also affected by the loss of BRCA1. These results might elucidate why mutations in BRCA1 causes cancer in the breast and ovaries, but not in other tissues.

Myriad Genetics Hordes Breast Cancer Data

Kathleen Sloan the president of the National Organization of Women has a troubling article at the Center for Bioethics and Culture website. It tells the story of a biotechnology company called Myriad Genetics and it BRCA1 & 2 test.

What the heck is BRCA1 & 2?  BRCA stands for “breast cancer” and mutations in BRCA 1 or 2 predispose females to breast and ovarian cancer. Mutations in BRCA genes also increase the risk of colon, prostate and pancreatic cancer.  Approximately 7% of breast cancer and 11 – 15% of ovarian cancer cases are caused by mutations in the BRCA genes.  If someone carries a mutation in either BRCA 1 or 2, they have a syndrome called Hereditary Breast and Ovarian Cancer (HBOC) syndrome.

The BRCA genes encode proteins that help repair DNA when it is damaged. Even though BRCA 1 & 2 work with several other proteins to accomplish this repair, mutations in the BRCA genes that compromise the quality of the proteins they encode can diminish the ability of cells to repair their DNA. Loss of efficient DNA repair systems leads to greater numbers of mutations in cells, some of which cause either loss of tumor suppress genes that normally put the brakes of cell proliferation, or activation of proto-oncogenes, which encode proteins that promote cell proliferation. Loss of tumor suppressor genes and activation of proto-oncogenes produces a cancer cell, and mutations in BRCA 1 or 2 and accelerate the onset of cancer cell formation (this is a highly simplified explanation and I apologize to the aficionados out there, but I am trying put the cookies on a nice low shelf).

Myriad Genetics came along and developed a genetic test for cancer-causing mutations in BRCA 1 & 2. This is good news, but Myriad Genetics is presently with holding their data from patients. This is not good news. Myriad Genetics wants to generate a database of mutations found in BRCA 1 and 2 genes from women all over the world. Some of these mutations do not affect the function of the encoded protein and do not predispose the patient to breast cancer, but some do. Which ones are harmful and which ones are not?

At this point things get sticky. Myriad has complied its sequence data on BRCA in order to construct a “variants of unknown significance” or VUS. Such a compilation would be invaluable, since it would help physicians correctly interpret the results of a breast cancer test. According to its present data archive, Myriad Genetics claims that only 3% of its tests fall into the VUS unknown category. However, other testing services report a 20% VUS rate. Who’s right? hard to say, given that Myriad Genetics will not release its data. Apparently they feel that their data has commercial value.

The problem is that lots of outfits that provided data to Myriad Genetics free of charge in order for them to develop their test. These other outfits have all their data available on public databases. What about Myriad Genetics – nope.


Myriad worked hard to develop this test – I do not think anyone is contesting that. Myriad Genetics has every right to make money off their test, but when they start hoarding potentially life-saving data, I think Ms. Sloan is right that they have crossed the line.

Myriad Genetics is also being sued because of their attempts to patent the BRCA genes. An impressive consortium of researchers, genetic counselors, women patients, cancer survivors, breast cancer and women’s health groups, and scientific associations representing 150,000 geneticists, pathologists and laboratory professionals are all plaintiffs in this lawsuit against the U.S. Patent Office, Myriad Genetics and the University of Utah Research Foundation, which hold the patents on the genes.

The lawsuit avers that patents on human genes violate the First Amendment because genes are “products of nature.” Therefore, such things cannot be patented. Such an argument has a strong intuitive appeal, and is almost certainly correct.

Read Ms. Sloan’s article here and see what you think.